Cellulitis is an infection of the dermis and subcutaneous fat that requires immediate medical care. It leads to nearly 500,000 US hospital admissions each year, many of which are re-admissions for recurrent or secondary infections. The repeated use of antibiotics in these patients drives up the expense of cellulitis therapy and contributes to the generation of antibiotic resistant bacteria strains. Further, some patients develop persistent lymphedema after cellulitis. Our preliminary data show that lymphatic pumping is impaired and lymph flow is reduced during and long after Staphylococcal aureus infections, a common and difficult to treat cause of cellulitis. Lymphatic dysfunction is associated with lymphedema formation and reduced immune function. We hypothesize that correcting lymphatic dysfunction after an initial infection will reduce the risk of both recurrent cellulitis and post-infection lymphedema. However, there are currently no FDA approved drugs indicated to improve lymphatic function. To develop a therapy to prevent lymphatic impairment, we first need to determine the mechanism by which S. aureus impairs lymphatic function. Our preliminary data show that S. aureus produces protein(s) that cause the death of lymphatic muscle cells, which are the cells responsible for lymphatic pumping that helps drive lymph flow. The lymphatic muscle cells do not recover after infection, leading to long term inhibition in lymphatic pumping. We have shown that a S. aureus strain with genetic deletion of the accessory gene regulator (agr)? which controls the production of many toxins?does not cause lymphatic dysfunction. Based on our strong preliminary data, we propose to use proteomic tools to identify the S. aureus products regulated by the agr that cause lymphatic muscle cell lysis in vitro (Aim 1). We will use mutant S. aureus strains that eliminate individual agr controlled gene products, as well as complement agr-/- S. aureus with overexpression of individual agr controlled gene products, to causally identify the responsible bacterial protein(s). We will then test the ability of mutant S. aureus strains with deletions of individual agr controlled gene products to alter lymphatic contraction, lymph flow and the duration of infection in vivo (Aim 2). These two exploratory Aims will allow us to define the mechanism of lymphatic impairment caused by S. aureus. Successful completion of this project will form the basis for exploring therapeutic strategies to restore lymphatic pumping and lymph flow after bacterial infections, with the long-term goals of preventing secondary infections and lymphedema, reducing hospital readmission rates, decreasing treatment costs and reducing the use of antibiotics. The expertise and facilities of the PI in studying lymphatic function, along with the expertise of our co-Investigator and scientific and clinical collaborators, provide optimal conditions for these innovative studies.